draft oecd test guideline determination of in vitro

DRAFT OECD TEST GUIDELINE 1

Determination of in vitro intrinsic clearance using cryopreserved rainbow trout 2

hepatocytes (RT-HEP) 3

4

INTRODUCTION 5

1. A major determinant of whether chemicals accumulate in fish is the extent to which 6

they undergo biotransformation. Standard test guidelines (e.g., OECD TG 305; 1) can be used 7

to measure chemical accumulation in fish, but these methods are expensive, time-consuming, 8

and require a substantial number of animals. It is critical, therefore, to have predictive 9

methods and models for bioaccumulation assessment available that are rapid, cost-effective, 10

and minimize the need for whole-animal testing. Ideally, such methods would be based on in 11

silico and/or in vitro approaches. 12

2. Nearly a decade ago, Nichols et al. (2) suggested that in vitro intrinsic clearance (CL, 13

IN VITRO, INT) rates, obtained using a substrate depletion approach, could be extrapolated to the 14

whole animal, and these in vivo clearance (CL, IN VIVO, INT) rates could be incorporated into 15

existing mass-balance models for fish bioconcentration factor (BCF) prediction. Since then, 16

several investigators have used in vitro systems derived from fish liver tissue to predict 17

biotransformation impacts on the accumulation of selected test chemicals (3-10). These 18

studies have shown that incorporating biotransformation estimates into BCF prediction 19

models substantially improves their performance; thus, predicted levels of accumulation are 20

much closer to measured values than are model predictions obtained assuming no 21

biotransformation. 22

3. This Test Guideline (TG) describes the use of cryopreserved rainbow trout 23

(Oncorhynchus mykiss) hepatocytes (RT-HEP) to determine the CL, IN VITRO, INT rate of a test 24

chemical using a substrate depletion approach. 25

4. Whole body biotransformation rate constants can be calculated using an appropriate in 26

vitro to in vivo extrapolation (IVIVE) model. These models use CL, IN VITRO, INT rates (derived 27

with this Test Guideline or Test Guideline RT-S9; 11) to estimate liver clearance rates, which 28

are then extrapolated to a whole-body (in vivo) biotransformation rate constant. An IVIVE 29

model applicable to rainbow trout was recently described by Nichols et al. (12). Crucial 30

parameters and use of IVIVE models are discussed in the OECD Guidance Document RT-31

HEP and RT-S9 (13) accompanying this Test Guideline and Test Guideline RT-S9 (11). 32

5. The results of recent ring trials involving three (10) and six laboratories (14) show that 33

RT-HEP can be used to reproducibly measure CL, IN VITRO, INT rates for chemicals covering a 34

range of physico-chemical properties. 35

6. Definitions of terms used in this document are provided in ANNEX 1. 36

INITIAL CONSIDERATIONS AND LIMITATIONS 37

7. The total incubation time should not exceed 4 h due to loss of viability of the RT-38

HEP. This limits the use of the test for chemicals metabolized at very low rates. The lowest 39

rate of in vitro activity which can be reliably quantified is approximately 0.05/h to 0.15/h (12, 40

15). More details are provided in (13). 41

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8. The reaction temperature should be at the acclimation temperature of the source fish. 42

Generally, rainbow trout are maintained at temperatures ranging from 10-15°C (e.g., the 43

incubation is carried out at 12°C if the source fish are maintained at 12°C). Since 44

biotransformation rates are temperature sensitive, the test temperature should be strictly 45

controlled at the acclimation temperature using a water bath, incubator, or thermomixer. 46

9. For volatile or otherwise difficult test chemicals, several alternative approaches are 47

suggested in the OECD Guidance Document RT-HEP and RT-S9 (13) such as use of tightly 48

closed GC vials or glass inserts test tubes (e.g., Hirschman test tubes) and passive dosing. 49

10. Before use of the Test Guideline on a mixture for generating data for an intended 50

regulatory purpose, it should be considered whether, and if so why, it may provide adequate 51

results for that purpose. Such considerations are not needed, when there is a regulatory 52

requirement for testing of the mixture. 53

11. The methodology as described here only measures depletion of a parent chemical. The 54

depletion approach could also be used to identify metabolites as described in OECD Guidance 55

Document RT-HEP and RT-S9 (13). 56

12. Hepatocytes from fish species other than rainbow trout could be used, provided that 57

primary hepatocytes can be successfully isolated and that protocols are adapted to species-58

specific considerations (see OECD Guidance Document RT-HEP and RT-S9; 13). 59

SCIENTIFIC BASIS OF THE METHOD 60

13. Rainbow trout provide a relatively easy source of hepatocytes, and the resulting RT-61

HEP have been shown to cryopreserve well, with minimal loss of xenobiotic metabolizing 62

capability (8, 9). This feature makes it possible to freeze RT-HEP in one location and 63

distribute them to other laboratories for later use, and to use one lot for several tests separated 64

in time. 65

14. Fresh primary cultured hepatocytes obtained from rainbow trout largely maintain their 66

epithelial phenotype, including functional glucose and lipid metabolism (16, 17), and the 67

activities of Phase I (e.g., cytochrome P450 (CYP)) and Phase II (e.g., sulfotransferases 68

(SULT), uridine 5’diphospho glucuronosyltransferases (UGT), glutathione transferases 69

(GST)) biotransformation enzymes (18). They possess functional membrane transporters, and 70

have been studied using well-known transporter substrates and inhibitors (19-22). 71

PRINCIPLE OF THE TEST 72

15. The CL, IN VITRO, INT rate of a test chemical is determined by using a substrate depletion 73

approach. The incubation system consists of RT-HEP (see ANNEX 2) suspended in Leibovitz 74

L-15 medium (L-15), typically at a cell density of 1-2 ×106 cells/mL. Introduction of the test 75

chemical to the RT-HEP suspension initiates the reaction. In order to collect samples at 76

various time points, the reaction is terminated by transferring an aliquot of the suspension to a 77

stopping solution. The decrease of the test chemical concentration from the reaction vial is 78

measured with a validated analytical method and used to determine the CL, IN VITRO, INT rate. 79

Incubations using enzymatically inactive RT-HEP are carried out as negative control to 80

distinguish between enzymatic biotransformation and abiotic decrease. 81

82

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INFORMATION ON THE TEST CHEMICAL 83

16. Before carrying out this test, the following information about the test chemical should 84

be known: 85

Solubility in water [TG 105; 23] 86

Solubility in cell culture medium 87

n-Octanol-water partition coefficient (Kow) or other suitable information on 88

partitioning behavior [TGs 107, 117, 123; 24-26] 89

Test chemical stability in water [TG 111; 27] and test medium 90

Vapor pressure [TG 104; 28] 91

Information on biotic or abiotic degradation, such as ready biodegradability [TGs 92

301, 310; 29, 30)] 93

Acid dissociation constant (pKa) for test chemicals that may ionize. 94

17. An appropriate analytical method, of known accuracy, precision, and sensitivity, for 95

the quantification of the test chemical in the test medium should be available, together with 96

details of sample preparation and storage. The analytical limit of quantification (LOQ) of the 97

test chemical in the test medium should be known. 98

REFERENCE CHEMICAL AND PROFICIENCY TESTING 99

18. It is recommended to use an appropriate reference chemical to verify the enzymatic 100

activity of the RT-HEP (also see §24 below). Points to be considered in choosing an 101

appropriate reference chemical are addressed in the OECD Guidance Document RT-HEP and 102

RT-S9 (13). 103

19. Reference chemicals can also be used to establish the test system in a laboratory. In an 104

inter-laboratory comparison, pyrene was used as reference chemical (14). 105

VALIDITY OF THE TEST 106

20. For a test to be valid, the following criteria should be met: 107

RT-HEP should be evaluated for the ability to catalyze Phase I and II metabolic 108

enzymatic reactions as described in ANNEX 3; 109

The pH of the recovery medium and L-15 must be adjusted to 7.8 ± 0.1; 110

Recovery of RT-HEP from cryopreservation should be ≥ 25%; 111

The cell density of the RT-HEP in suspension should be within 25% of its target 112

density (e.g., 2.0 ± 0.5 x 106

cells / mL); 113

Cell viability in the RT-HEP suspension should be ≥ 80%; 114

The incubation temperature should be at the acclimation temperature of the 115

source fish (e.g., 10-15°C); 116

Negative (enzymatically inactive RT-HEP) controls should demonstrate no 117

significant loss of parent chemical over the incubation time (e.g., < 20% of loss 118

determined in active RT-HEP preparations); 119

A minimum of six time points should be used to determine the CL, IN VITRO, INT 120

rate, i.e. to calculate the regression and derive the slope, with an R2 value >0.85. 121

In the case of chemicals that are very slowly metabolized (e.g., a very shallow 122

slope), the R2 may not be ≥0.85. In this instance, careful consideration should be 123

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given to whether the slope is significantly different than zero before including or 124

excluding the run. 125

A minimum of two independent runs must be performed (see §30). If the 126

calculated regression from the two runs with active RT-HEP are significantly 127

different (e.g., t-test of the slopes with p<0.05), then a third run should be 128

performed. 129

130

DESCRIPTION OF THE METHOD 131

Apparatus 132

21. The following equipment is required for RT-HEP incubation: 133

4°C refrigerator; 134

-20°C freezer; 135

Liquid nitrogen storage tank or ultralow temperature freezer (-150°C) for 136

storage of cryopreserved RT-HEP; 137

Analytical balance to measure reagents and test chemicals; 138

pH meter; 139

Vortex mixer; 140

Refrigerated centrifuge for 50 mL tubes; 141

50 mL conical centrifuge tubes; 142

Sample incubation equipment, e.g., shaking water bath with chiller, shaking 143

incubator with heating and cooling functions, or thermomixer block with 144

shaking capabilities; 145

Glass ware for preparing solutions, reagents, etc; 146

Glass vials for incubation test (e.g., 7 mL scintillation test tubes); 147

1.5 mL micro-centrifuge tubes; 148

Small benchtop refrigerated centrifuge for micro-centrifuge tubes; 149

Sample glass vials for HPLC/GC or other analytical instruments; 150

Pipettes and tips; 151

Appropriate equipment and reagents for the counting of RT-HEP. 152

Chemicals for analytical measurements 153

22. The following chemicals are required: 154

Solvent to dissolve test chemical, analytical grade or equivalent (e.g., 155

methanol, acetonitrile, acetone); the solvent must be miscible with the aqueous 156

media used in the RT-HEP suspension; 157

Stopping and extraction solvents, analytical grade or equivalent (e.g., 158

methanol, acetonitrile, methylene chloride, methyl tert-butyl ether). 159

Cryopreserved RT-HEP 160

23. Cryopreserved RT-HEP can be obtained from commercial sources, if available, or 161

prepared as described in ANNEX 2. 162

24. Each RT-HEP lot should be evaluated for the protein concentration (see ANNEX 2) 163

and its ability to catalyze Phase I and II biotransformation reactions (see ANNEX 3). 164

5

Standardized assays to determine EROD activity (CYP1A), glutathione transferase activity 165

(GST) and uridine 5'-diphospho-glucuronosyltransferase (UGT) activity are briefly described 166

in ANNEX 3. These characterization assays or a known reference chemical should be used to 167

test a new lot of RT-HEP at the beginning of the study or before the lot is used for the first 168

time. They should also be used occasionally to monitor possible activity losses during 169

storage. 170

25. The inclusion of enzymatically inactive RT-HEP is mandatory. Incubations using 171

inactive RT-HEP serve as a negative control to distinguish between enzymatic 172

biotransformation and other potential loss processes, such as adsorption to the reaction vial, 173

volatilization, and abiotic degradation. A protocol for enzyme inactivation by heating is 174

provided in ANNEX 4. Other methods to inactivate the RT-HEP can be used, but were not 175

explored in the ring trial (14). 176

Cell culture medium and reagents 177

26. Cell culture medium and reagents needed for thawing and incubation of RT-HEP are 178

included in ANNEX 5. 179

Test set-up 180

27. Preliminary experiments that include range finding conditions (e.g., test chemical 181

concentration, RT-HEP concentration and incubation time) should be conducted to establish 182

reaction conditions needed to reliably measure intrinsic in vitro hepatic clearance of the test 183

chemical. ANNEX 6 details how conditions that result in first-order depletion kinetics can be 184

determined. 185

28. A sufficient number of sampling time points should be obtained to develop a high-186

quality regression of log-transformed chemical concentration data. At least six time points 187

should be used to generate this regression. 188

29. An example test set-up using a single vial approach with seven time points is shown in 189

Figure 1 of ANNEX 7. This test set-up is recommended to test chemicals that are not 190

difficult-to-test (e.g., non-volatile, do not bind to vessel walls, and distribute rapidly through 191

the reaction system) at one test concentration. It generally produces the least variable results 192

and is simplest to perform. For volatile test chemicals, the multiple vial approach is 193

recommended (ANNEX 7; Figure 2). 194

30. Each test consists of at least two independent runs to determine the CL, IN VITRO, INT 195

rate. Each independent run is performed on a different day or on the same day provided that 196

for each run: a) independent fresh stock solutions and working solutions of the test chemical 197

are prepared and b) independently prepared RT-HEP suspensions are used. If the calculated 198

regression from the two runs with active RT-HEP are significantly different (e.g., t-test of the 199

slopes with p<0.05), then a third run should be performed. 200

31. For each run, one replicate each for active RT-HEP and enzymatically inactive RT-201

HEP is spiked with the test chemical, and one replicate for active RT-HEPs is spiked with a 202

reference chemical. Samples are collected at each time point (e.g., 5, 10, 15, 20, 40, 60, 90 203

min). In some cases, more replicates may be required to ensure accuracy of the analytical 204

method for the given test chemical. 205

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32. In the following, the single vial approach is described whereas the multiple vial 206

approach is detailed in ANNEX 7. 207

Preparation of test chemical, media and stopping solutions 208

33. Stock solution(s) of the test chemical should be prepared in reaction buffer (L-15 209

medium) or an adequate solvent that is previously tested. Typical solvents include acetone, 210

acetonitrile, and methanol. The stability of the test chemical in the stock solution should be 211

evaluated in advance of the test if stock solutions are not prepared daily (see 13). 212

34. On the day of the test, the desired spiking concentration of the test chemical is 213

prepared by diluting the stock solution with reaction buffer (L-15 medium) or an organic 214

solvent based on the results of the preliminary experiments (see §27; ANNEX 6; 13). If an 215

organic solvent is used, the total amount in the incubation RT-HEP suspension should be kept 216

as low as possible and not exceed 1% to avoid suppressing enzyme activity. In general, the 217

test chemical concentration should be as low as possible (according to the LOQ of the 218

analytical method) and result in first-order depletion kinetics as determined in the preliminary 219

experiments (§27). 220

35. A stopping solution (e.g., methanol, acetonitrile, methylene chloride, methyl tert-butyl 221

ether) is prepared which may include an internal standard. For most tests, 1.5 mL micro-222

centrifuge tubes may be filled with the stopping solution in advance (e.g., 100 µL sample 223

terminated in 400 µL stopping solution) and stored on ice. For volatile solvents (e.g., solvents 224

that vaporize at room temperature, such as methylene chloride, methyl tert-butyl ether), the 225

tubes should remain capped and kept cool, or the solvents should be added directly prior to 226

collection of the time point. For solvents which interact with plastic, glass tubes may be used 227

to stop the reactions (see also OECD Guidance Document RT-HEP and RT-S9, 13). 228

Preparation of RT-HEP suspension 229

36. The volume of RT-HEP suspension (active or enzymatically inactive) required for 230

each reaction vial depends on the number of desired time points as well as the volume to be 231

removed for each time point. To ensure good mixing at the final time point, the volume of the 232

RT-HEP incubation remaining should be still large enough to cover the bottom of the vial. 233

37. For the single vial approach (Figure 1, ANNEX 7), two vials with active and one vial 234

with enzymatically inactive RT-HEP suspensions are used per run. In preparing the two 235

suspensions, an additional 25% is recommended to provide a modest excess of biological 236

material, taking into account the number of time points in the test set-up. For example, if 1.0 237

mL per vial will be used in the test, a total of about 2.5 mL (2.0 mL + 25%) should be 238

prepared for the active RT-HEP suspension and 1.25 mL (1.0 + 25%) for the enzymatically 239

inactive RT-HEP suspension. 240

38. Incubations are typically conducted using 1-2 x 106 viable hepatocytes/mL. RT-HEP 241

counting should be performed using a basic methodology as described in Fay et al. (31). After 242

thawing (ANNEX 5), the number of viable cells is determined and adjusted to the desired cell 243

density in L-15 medium, pH 7.8. To obtain highly accurate estimates of the RT-HEP cell 244

density, additional cell counts may be performed on the RT-HEP suspension once it has been 245

diluted to its reaction concentration. Three additional cell counts (post-dilution) are 246

recommended (using separate dilutions with trypan blue) on the reaction suspension. This 247

second set of counts may be performed during or after the incubations as time allows. If the 248

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three cell counts vary by more than 20 % CV, an additional cell count may be needed to 249

obtain an accurate estimation of the active RT-HEP cell density. 250

39. The desired volume (e.g., 1 mL for seven time points) of active and enzymatically 251

inactive RT-HEP suspension is added to the respective reaction vials. After loosely capping 252

the vials, they are pre-incubated at the test temperature (e.g., 10-15°C) for 10 min with gentle 253

shaking. RT-HEP should not be shaken vigorously or vortexed. 254

Incubation with test chemical and stopping of reaction 255

40. Test chemical (usually 5μL; however, this depends on the concentration of the spiking 256

solution) is directly added into the suspension of each reaction vial to initiate the reaction. 257

The vials are swirled to distribute the chemical and loosely capped. Throughout the 258

incubation, RT-HEP should be gently shaken as above (§39) at the test temperature (e.g., 10-259

15°C). 260

41. For sampling at a specified time point, the reaction vial is removed from the water 261

bath or incubator, gently swirled or shaken to form a homogenous suspension, and an aliquot 262

(e.g., 100 µL) is removed with a pipette and directly dispensed into the corresponding 1.5 mL 263

micro-centrifuge tube containing ice-cold stopping solution kept on ice (see §35). To ensure 264

quantitative transfer of the sample, pipetting up and down in the solvent three times is 265

recommended. 266

42. The micro-centrifuge tubes are kept on ice until samples from all time points have 267

been collected. It may be useful to refrigerate samples overnight to facilitate complete protein 268

precipitation prior to centrifugation if a water miscible solvent is used as stopping solution. If 269

volatile solvents like methylene chloride, and methyl tert-butyl ether are used, the samples 270

must be extracted if possible directly after stopping the reaction. Preliminary experiments 271

should be performed to confirm complete precipitation of proteins upon termination of the 272

reaction (ANNEX 6). 273

43. After the sampling is completed or for volatile solvents at each sampling point, micro-274

centrifuge tubes are vortexed (e.g., for 3 min at 1500 to 2000 rpm) and centrifuged (e.g., 15 275

min at 20,000 g and 4°C). Some test chemicals may require overnight refrigeration to ensure 276

maximal extraction with the solvent. Additional considerations are provided in OECD 277

Guidance Document RT-HEP and RT-S9 (13). The supernatant is transferred to analytical 278

HPLC/GC sample vials and stored at -20°C until analysis. 279

ANALYTICAL MEASUREMENTS 280

44. The concentration of the test chemical is determined using a validated analytical 281

method. More details are provided in OECD Guidance Document RT-HEP and RT-S9 (13). 282

45. Since the whole procedure is governed essentially by the accuracy, precision and 283

sensitivity of the analytical method used for the test chemical, check experimentally that these 284

qualities of the chemical analysis, as well as recovery (e.g., 80%) of the test chemical from 285

the test medium are satisfactory. 286

DETERMINATION OF IN VITRO INTRINSIC CLEARANCE 287

46. The log10-transformed substrate concentrations are plotted against time and should 288

demonstrate a log-linear decline (R2 value ≥0.85). 289

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47. If a visual inspection of the regression shows obvious outliers, a statistically valid 290

outlier test may be applied to remove spurious data points (e.g., as described in [26]) as well 291

as documented justification for their omission. In some cases, non-linear behavior may be 292

observed at the beginning or end of a test, which could be due to problems with dissolution of 293

the test chemical or loss / inhibition of enzyme activity. However, the depletion rate should 294

be determined from the linear portion of the curve, with a minimum of six data points. 295

296

48. A first-order elimination rate constant, ke (1/h), is determined as -2.3 × slope of the 297

log-linear decline. 298

49. ke is divided by the measured viable (e.g., as determined by trypan blue exclusion) 299

RT-HEP concentration to obtain the CL, IN VITRO, INT (mL/h/106 cells) rate. If RT-HEP were 300

counted pre- and post-dilution of RT-HEP suspension, the post-dilution counts should be 301

used. 302

TEST REPORT 303

50. The test report should include the following: 304

Test chemical 305

Mono-constituent substance: 306

o physical appearance, water solubility, and additional relevant physicochemical 307

properties; chemical identification, such as IUPAC or CAS name, CAS 308

number, eSMILES or InChI code, structural formula, purity, chemical identity 309

of impurities as appropriate and practically feasible, etc. 310

Multi-constituent substance, unknown or variable composition, complex reaction 311

products or of biological materials (UVCBs) and mixtures: 312

o characterized as far as possible by chemical identity (see above), quantitative 313

occurrence and relevant physicochemical properties of the constituents. 314

Analytical method for quantification of the test chemical 315

Rainbow trout hepatocytes 316

If purchased: 317

o Commercial source 318

o Rainbow trout supplier 319

o Rainbow trout strain 320

o Acclimation temperature 321

o Fish weight 322

o Liver weight 323

o Gonadosomatic index (for determination of sexual maturity) 324

If prepared in-house, see reporting template in ANNEX 2 325

Characterization (see ANNEX 3) 326

Recovery rate after thawing 327

Viability of RT-HEP in suspension 328

Test conditions 329

Concentration of test chemical and reference chemical 330

9

Method of preparation of stock solution(s) of test chemical (name and concentration of 331

solvent, if applicable) 332

Cell culture medium characteristics (temperature, pH) 333

Incubation temperature 334

RT-HEP cell density in suspension for reaction 335

Test set-up (single vial or multiple vial approach) 336

Number of replicates (if more than one is used per run) 337

Number of independent runs 338

Incubation temperature 339

Time points 340

Description of preliminary experiments 341

Analytical method 342

Method used (recovery efficiency, limit of quantification) 343

Matrix used for standard preparation 344

Internal standard 345

Statistical method 346

Description and statistical method used for exclusion of time points and/or runs. 347

Results 348

Results from any preliminary experiment performed 349

Values from individual vials, time points for each independent run 350

Complete description of all chemical analysis procedures employed including limits of 351

detection and quantification, variability, and recovery 352

Calculated CL, IN VITRO, INT rates from independent incubations with active and 353

enzymatically inactive RT-HEP 354

Average and standard deviation values from independent runs, as well as results from 355

t-tests to compare average CL, IN VITRO, INT from the runs. 356

Any excluded time points or runs 357

Anything unusual about the test, any deviation from the test guideline and any other 358

relevant information 359

360

REFERENCES 361

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OECD Guidelines for the Testing of Chemicals, Section 3, OECD Publishing. doi: 363

10.1787/9789264185296-en. 364

2. Nichols, J.W., Schultz, I.R., and Fitzsimmons, P.N. 2006. In vitro-in vivo extrapolation of 365

quantitative hepatic biotransformation data for fish: I. A review of methods, and strategies 366

for incorporating intrinsic clearance estimates into chemical kinetic models. Aquatic 367

Toxicology 78:74-90. 368

3. Cowan-Ellsberry, C.E., Dyer, S.D., Erhardt, S., Bernhard, M.J., Roe, A.L., Dowty, M.E., 369

and Weisbrod, A.V. 2008. Approach for extrapolating in vitro metabolism data to refine 370

bioconcentration factor estimates. Chemosphere 70:1804-1817. 371

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intrinsic clearance in freshly isolated hepatocytes from rainbow trout (Oncorhynchus 381

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281:R1119-R1126. 432

22. Žaja, R., Munić, V., Klobučar, R.S., Ambriović-Ristov, A., and Smital, T. 2008. Cloning 433

and molecular characterization of apical efflux transporters (ABCB1, ABCB11 and 434

ABCC2) in rainbow trout (Oncorhynchus mykiss) hepatocytes. Aquatic Toxicology 435

90:322-332. 436

23. OECD 1995. Test No. 105: Water Solubility, OECD Guidelines for the Testing of 437

Chemicals, Section 1, OECD Publishing. doi: 10.1787/9789264069589-en. 438

24. OECD 1995. Test No. 107: Partition Coefficient (n-octanol/water): Shake Flask Method, 439

OECD Guidelines for the Testing of Chemicals, Section 1, OECD Publishing. doi: 440

10.1787/9789264069626-en. 441

25. OECD 2006. Test No. 123: Partition Coefficient (1-Octanol/Water): Slow-Stirring 442

Method, OECD Guidelines for the Testing of Chemicals, Section 1, OECD Publishing. 443

doi: 10.1787/9789264015845-en. 444

26. OECD 2004. Test No. 117: Partition Coefficient (n-octanol/water), HPLC Method, OECD 445

Guidelines for the Testing of Chemicals, Section 1, OECD Publishing. doi: 446

10.1787/9789264069824-en. 447

27. OECD 2004. Test No. 111: Hydrolysis as a Function of pH, OECD Guidelines for the 448

Testing of Chemicals, Section 1, OECD Publishing. doi: 10.1787/9789264069701-en. 449

28. OECD 2006. Test No. 104: Vapour Pressure, OECD Guidelines for the Testing of 450


29. OECD 1992. Test No. 301: Ready Biodegradability, OECD Guidelines for the Testing of 452


30. OECD 2006. Test No. 310: Ready Biodegradability - CO2 in sealed vessels (Headspace 454

Test), OECD Guidelines for the Testing of Chemicals, Section 3, OECD Publishing. doi: 455

10.1787/9789264016316-en. 456

31. Fay, K.A., Nabb, D.L., Mingoia R.T., Bischof, I., Nichols, J.W., Segner, H., Johanning, 457

K., and Han, X. 2015. Determination of metabolic stability using cryopreserved 458

hepatocytes from rainbow trout (Oncorhynchus mykiss). Current Protocols in Toxicology 459

65:4.42.1-29. 460

32. OECD 2006. OECD Series on Testing and Assessment No. 54. Current approaches in the 461

statistical analysis of ecotoxicity data: A guidance to application. Available on: 462

http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?doclanguage=en&cot463

e=env/jm/mono%282006%2918. 464

465

http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?doclanguage=en&cote=env/jm/mono%282006%2918

http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?doclanguage=en&cote=env/jm/mono%282006%2918

12

ANNEX 1 466

ABBREVIATIONS & DEFINITIONS 467

468

BCF Bioconcentration factor (L/kg) 469

BSA Bovine serum albumin 470

CL, IN VITRO, INT in vitro intrinsic clearance (mL/h/106 cells or mL/h/mg 471

protein) 472

CL, IN VIVO, INT in vivo intrinsic clearance (ml/h/g fish) 473

CV Coefficient of variation 474

CYP Cytochrome P450 475

DMSO Dimethyl sulfoxide 476

DMEM Dulbecco's Modified Eagle Medium 477

EDTA Ethylenediaminetetraacetic acid 478

EROD Ethoxyresorufin-O-deethylase 479

FBS Fetal bovine serum 480

First-order depletion kinetics A chemical reaction in which the rate of decrease in the 481

number of molecules of a substrate is proportional to the 482

concentration of substrate molecules remaining 483

GC Gas Chromatography 484

GSH L-Glutathione 485

GSI Gonadosomatic index 486

GST Glutathione transferase 487

HBSS Hanks' Balanced Salt Solution 488

HPLC High Performance Liquid Chromatography/ 489

IVIVE model In vitro to in vivo extrapolation model 490

ke Elimination rate constant 491

KM Michaelis-Menten constant 492

Kow n-Octanol-water partition coefficient 493

L-15 Leibovitz-15 494

13

LOQ Limit of quantification 495

MS-222 Tricaine methanesulfonate 496

pKa Acid dissociation constant 497

rpm Revolutions per minute 498

RT-HEP cryopreserved rainbow trout hepatocytes 499

SULT Sulfotransferase 500

TG Test Guideline 501

UGT Uridine 5'-diphospho-glucuronosyltransferase 502

Vmax Maximum enzymatic rate at saturating test chemical 503

concentration 504

505

14

ANNEX 2 506

ISOLATION AND CRYOPRESERVATION OF RAINBOW TROUT HEPATOCYTES 507

(RT-HEP) 508

FISH 509

1. RT-HEP should be preferably obtained from sexually immature rainbow trout since 510

previous work has shown that sexually immature rainbow trout (Oncorynchus mykiss) do not 511

differ with respect to their metabolic capabilities in relation to their gender (1-3). RT-HEP 512

can therefore be collected without regard to gender. 513

2. If fish are obtained from a supplier, they should be acclimatized in the laboratory for 514

at least 2 weeks prior to use. Fish should not receive treatment for disease in the two-week 515

acclimation period and any disease treatment by the supplier should be completely avoided if 516

possible. Fish with clinical signs of disease should not be used. 517

3. Rainbow trout are typically raised at 10-15ºC. The temperature of the holding tank in 518

the laboratory should be similar and maintained at ± 2°C. Holding density of fish should be 519

low enough to ensure optimal growth and welfare. 520

4. Measure and record water chemistry characteristics at periodic intervals, including: 521

pH, total alkalinity (as mg/L CaCO3), dissolved oxygen (mg/L, converted to percent 522

saturation), and total ammonia (mg/L). 523

5. Record fish maintenance details as well, including: photoperiod, feeding regime, feed 524

type, water temperature, holding density (kg fish/liter tank volume), and number of fish/tank. 525

This specific information should be reported to allow for isolation-specific parameters to be 526

used in subsequent applications, such as BCF prediction models. 527

PROCEDURE SUMMARY 528

6. The procedure for isolating hepatocytes from rainbow trout largely follows techniques 529

used to obtain hepatocytes from mammals (4-8). 530

7. The fish is anesthetized (but alive) during the isolation procedure to take advantage of 531

the dilated hepatic vascular system, which allows for increased perfusion efficiency. The 532

hepatic portal vein is cannulated, and the liver is perfused with a Ca2+

/Mg2+

-free balanced salt 533

buffer (Buffer I; see Table 1) to clear the liver of blood and loosen desmosomes. The liver is 534

then perfused by a balanced salt solution (with Ca2+

and Mg2+

) containing the enzyme 535

collagenase (Buffer II; see Table 1). After digestion, the collagenase reaction is terminated by 536

perfusion with cell medium containing bovine serum albumin (Buffer III; see Table 1), and 537

the hepatocytes are mechanically separated from the liver capsule. 538

8. The primary hepatocytes are washed and purified using a density gradient. All buffers 539

are adjusted to the pH of the fish’s blood and chilled to its acclimation temperature. For 540

rainbow trout, this is typically pH 7.8 at 12°C. 541

9. Generally, pooling hepatocytes from several fish (three to six) is recommended. This 542

approach will diminish the influence of a single fish, and better represent a population. 543

Elapsed time from isolation to use, or cryopreservation, should be minimized. Quickly 544

isolating hepatocytes from several fish may be accomplished by operating several isolation 545

15

stations simultaneously, or by splitting the perfusate line to accommodate more than one fish. 546

This latter scenario is described below. 547

APPARATUS AND MATERIAL 548

10. Apparatus 549

Vessels to expose fish to anesthetic 550

Digital balance (1 g – 2000 g), weigh boats 551

Refrigerated centrifuge (e.g., for 50 mL tubes) 552

Conical centrifuge tubes, e.g., 50 mL 553

Forceps, large and small sharp surgical scissors 554

Mesh (e.g., 100 µm) 555

Tall glass beaker, 150 mL 556

21G × ¾ safety winged infusion set (butterfly catheter) 557

Micro-bulldog clamps (optional; Harvard Apparatus, NP 52-3258) 558

Perfusion apparatus (Figure 3) including: 559

o Recirculating water bath capable of chilling water to 12°C 560

o Peristaltic pump 561

o Pump tubing 562

o Water-jacketed glass coil condenser (45 mm × 260 mm or similar) 563

o Water-jacketed glass bubble trap, with stopcock 564

o Surgical platform with catch basin for blood and perfusate (optional; a tray 565

lined with paper toweling is also sufficient) 566

Serological pipets and pipetman 567

Buckets with ice 568

Cryogenic container for cell storage 569

Liquid nitrogen 570

Equipment for counting cells 571

11. Buffers, cell culture media, chemicals 572

Tricaine Methanesulfonate (MS-222) 573

Sodium bicarbonate (NaHCO3) 574

Ethanol, 70% (v/v) 575

Percoll 576

Hydrochloric acid (HCl), 1 N 577

Leibovitz L-15 medium (L-15) 578

Hanks’ Balanced Salt Solution (HBSS) 579

Ethylenediaminetetraacetic acid disodium salt (EDTA) 580

Bovine Serum Albumin (BSA) 581

Fetal Bovine Serum (FBS) 582

Dimethyl sulfoxide (DMSO) 583

Dulbecco’s Modified Eagle Medium (DMEM) 584

Collagenase (type IV) 585

586

587

16

PREPARATION OF REAGENTS AND SOLUTIONS 588

12. The tricaine methanesulfonate (MS-222; 150 mg/L) should be prepared with water 589

from the same source used to maintain the fish prepared. For example, for 8 L, 1.2 g MS-222 590

is added to the water and mixed until dissolved. A predetermined amount of NaHCO3 is used 591

to maintain the source water pH. If the water is low-alkalinity, the required mass of NaHCO3 592

is approximately 3 times that of the MS-222. 593

13. The three perfusion buffers are prepared as listed in Table 1. The amounts provided 594

are sufficient to perfuse three to four fish. 595

Table 1: Perfusion buffers I, II, and III 596

Reagent Per 600 mL preparation Concentration

Buffer I 1 × HBSS (without Ca2+

/Mg2+

salts) 600 mL

pH 7.8 EDTA 510 mg 2.3 mM

NaHCO3 212 mg 4.2 mM

Buffer II 1 × HBSS (with Ca2+

/Mg2+

salts) 600 mL

pH 7.8 Collagenase, type IV 150 mg** 0.25 mg/mL**

NaHCO3 212 mg 4.2 mM

Buffer III DMEM 600 mL

pH 7.8 BSA 6.0 g 1% (w/v)

** Buffer II: Collagenase activity varies from lot to lot, and is not a pure preparation of enzyme, but contains 597 other proteases, polysaccharidases, and lipases. It may be necessary to adjust the amount used depending on how 598 well the liver digests. 599

14. The 90% Percoll solution for cell purification should be prepared using a bio-hood 600

and sterile technique. The temperature of the Percoll should match the test conditions (the 601

temperature at which the fish are acclimated). 90 mL of chilled Percoll is added to a 602

graduated cylinder and the volume adjusted up to 100 mL with DPBS 10 × solution. After 603

mixing well, the solution is adjusted to pH 7.8 by slowly adding 1 N HCl. If the pH drops 604

below 7.8, NaOH should not be added since it forms a precipitate and turns the solution 605

cloudy. Instead, additional Percoll/DPBS should be added to increase the pH. The solution 606

should be stored at 2-8°C for up to 14 d. 607

15. The cryopreservation medium may be prepared the day before use, but the pH should 608

only be adjusted 1-2 h prior to hepatocyte isolation. The ingredients are provided in Table 2. 609

The pH may need to be adjusted to fully dissolve the albumin. Allow the solution to sit 610

overnight at 1-10°C to reduce foam. On the day of use, adjust the buffer pH to 7.8 at 12°C 611

and sterile filter through a 0.2 µm polyethersulfone filter. 612

16. Table 2 further indicates how cryopreservation medium with 12% and 16% DMSO, 613

respectively, are prepared. 614

615

17

Table 2. Cryopreservation medium recipes 616

Reagent Per 200 mL preparation Concentration

Cryopreservation

buffer

pH 7.8 at 12°C

DMEM 160 mL

FBS 40 mL 20% (v/v)

BSA 0.5 g 0.25% (w/v)

Cryopreservation

medium with 12%

DMSO

Combine 1.8 mL of DMSO for every 13.2 mL cryopreservation

buffer

Cryopreservation

medium with 16%

DMSO

Combine 7.2 mL of DMSO for every 37.8 mL cryopreservation

buffer

617

DETAILED DESCRIPTION 618

Preparation of apparatus and buffers on the day of RT-HEP isolation and 619

cryopreservation 620

Apparatus 621

17. A recommended set up for the perfusion apparatus is shown in Figure 1. Alternate 622

set-ups may also be used (e.g., peristaltic pump). The perfusate flow is set to approximately 623

10 mL/min. Note that the flow rate may need adjustment for fish of different sizes (e.g., 5 624

mL/min for 100 g fish). 625

18. If perfusing two fish with one apparatus, the line exiting the bubble trap may be split. 626

A clamp or valve should be used to control the perfusate flow through the second line while 627

starting perfusion on the first fish. In this case, the pump rate needed for 10 mL/min flow 628

through in the first line when the second is clamped should be determined and the necessary 629

increase in pump rate required to maintain the flow rate when both perfusion lines are open. 630

19. The temperature of perfusate exiting the cannula should be approximately 12°C (or the 631

temperature at which the fish was acclimated) and the temperature setting of the chiller and 632

circulator should be adjusted accordingly. 633

20. Prior to the start of the liver perfusion (see §33), the tubing and bubble trap of the 634

perfusion apparatus is flushed with 70% ethanol for approximately 10 min, followed by 10 635

min flushing with distilled water, and finally with perfusion buffer I for 3 min. 636

21. To flush the bubble trap, open the top and front valves to empty. With the front valve 637

closed, fill the bubble trap with fluid until it spills out the top valve. Next, discharge the 638

majority of the fluid by releasing the front valve. Flush in this manner several times. 639

22. For filtering the hepatocytes suspension, a piece of nylon mesh (e.g., 100 μm) is 640

secured over the rim 125 mL tall glass beaker using a rubber band. The beaker should be 641

placed on ice. The mesh should not be tight across the top of the beaker, but depressed in the 642

center to filter the RT-HEP suspension. Note that poor quality nylon may not be sufficient for 643

use in filtering hepatocytes. Stitching with ragged edges may damage hepatocytes. 644

18

23. All surgical supplies should be set out, including: forceps, large and small scissors, 645

weigh boat, butterfly catheter set, and micro-bulldog clamp or sutures. 646

Perfusion buffers and cryopreservation media 647

24. Within 2 h prior to use, the pH of pre-prepared perfusion buffers should be adjusted to 648

the target pH at the acclimation temperature of the fish (e.g., pH 7.8 at 12°C), if necessary. 649

Perfusion buffers I and II, especially, will decrease in pH if prepared too far in advance due to 650

the dissolution of CO2 and formation of carbonic acid. 651

Preparation of fish and surgery 652

25. Fish should fast 24 h prior to isolation of hepatocytes. 653

26. Using a net, fish are captured and transferred to a tank or bucket containing 8 L of 654

anesthetic solution (MS-222) prepared earlier. The MS-222 solution can be used to 655

anesthetize several fish without loss of anesthetic efficacy, but this number may depend on 656

the size of the fish. 657

27. The fish should be immersed in the MS-222 solution for at least 1 min. The fish is 658

properly anesthetized when opercular movement has ceased, there is a total loss of 659

equilibrium and muscle tone, and no response to stimuli (a firm squeeze at the base of the tail 660

may be used to determine response to stimuli). After finalisation of the liver perfusion (§§ 34-661

36), the fish should be humanely killed with a sharp blow on the head. 662

28. The weight and length of anesthetized fish are recorded (see Reporting Template). 663

29. The fish is placed on the surgical platform with the ventral surface facing the 664

technician. As illustrated in Figure 2, the following incisions are recommended: a) midline 665

incision from the vent to the isthmus, taking care not to cut too deeply into the body cavity; 666

followed by b) a lateral incision at the caudal end of the midline incision extending about half 667

way up to the dorsal surface and c) a similar lateral incision just caudal to the operculum. 668

30. By folding back and cutting away the flap of tissue resulting from the incisions 669

described above, the body cavity is exposed and the liver should be dark red and the heart 670

should still be beating (Figure 3). The ventral branch of the hepatic portal vein (running from 671

the intestine to the liver hilus) should be located and carefully cleared from any obscuring 672

connective tissue. 673

31. The perfusate pump set to 10 mL/min is turned on. If perfusing 2 fish with one 674

apparatus, the second perfusion line should be clamped initially. Both fish should be prepared 675

to the point where they are ready for liver perfusion: anesthetized, weighed, body cavity 676

exposed, and hepatic portal veins located. 677

32. With perfusion buffer I flowing, a 21-G butterfly catheter is carefully inserted into the 678

portal vein in the direction of the liver, and secure in place with a micro-bulldog clamp 679

(Figure 4). Different gauge catheters may be preferred depending upon the size of the fish. If 680

the micro-bulldog clamp is not available, sutures or pressure applied by fingers may be 681

substituted, as described in (2). 682

33. The blood vessels leading from the anterior aspect of the liver to the heart are severed, 683

or, alternatively, the heart may be severed or removed completely to allow efflux of perfusate 684

19

(Figure 4). If perfusing 2 fish from one apparatus, the second perfusion line should be 685

unclamped just prior to cannulating the portal vein of the second fish, and the pump rate 686

increased to provide the target perfusion flow rate (e.g., 10 mL/min) in both perfusion lines. 687

The liver of the first fish is perfused with perfusion buffer I while the second fish is prepared 688

(approximately 1 min). 689

Liver Perfusion 690

34. Liver perfusion is started with perfusion buffer I for 8-12 min. Blanching of the liver 691

should be evident within the first minute of perfusion (Figure 4). It is followed by perfusion 692

buffer II for 12-15 until the liver visibly softens. After approximately 5 min of perfusion with 693

perfusion buffer II, the liver may periodically be prodded gently with blunt forceps to test for 694

softening. Generally, perfusion with perfusion buffer II beyond 15 min will result in over-695

digestion and is not recommended. 696

35. Once the liver has softened sufficiently, the collagenase digestion is terminated by 697

perfusing for 3 min with perfusion buffer III. 698

36. Switching perfusates may be accomplished by quickly transferring the draw tubing to 699

the reservoir containing the next buffer, or by using a split line with a valve on the draw 700

tubing to switch between buffers. The inclusion of a bubble trap in the perfusion apparatus 701

will prevent any air introduced to the perfusion line during the buffer transition from reaching 702

the liver. 703

RT-HEP isolation 704

37. The flow of perfusion buffer III is stopped and the catheter removed. Using small, 705

sharp scissors, the liver is excised along with the intact gall bladder. The gall bladder is 706

carefully cut away the without rupturing and the liver transferred to a weigh boat containing 707

~30 mL of ice-cold perfusion buffer III (Figure 5). If the gall bladder ruptures during this 708

process, the liver should be rinsed with perfusion buffer III to remove any bile prior to its 709

transfer to the weigh boat containing perfusion buffer III. 710

38. Using sharp forceps or the ends of small, sharp scissors, the Glisson's capsule is torn 711

open and the liver gently shaken in perfusion buffer III to release the hepatocytes (Figure 6). 712

The liver may be gently raked with forceps or scissors to facilitate recovery of hepatocytes. 713

The scraping and gentle shaking of the liver capsule may take several minutes to collect a 714

sufficient number of hepatocytes. 715

39. The crude hepatocyte suspension is filtered through the nylon mesh and hepatocytes 716

are collected in the beaker (Figure 7) prepared as indicated in §22. The remaining liver 717

connective tissue may be gently pushed against the mesh to increase hepatocyte recovery; 718

however, excessive handling will produce poorer quality hepatocytes (e.g., caused by 719

formation of blebs). 720

40. The mesh should be primed with a small amount of perfusion buffer III prior to 721

pouring the hepatocytes to minimize initial sheer stress. Alternatively, nylon mesh tube 722

inserts may be purchased for use with 50 mL conical tubes. These tube inserts should also be 723

primed with a small amount of perfusion buffer III. 724

20

41. Prior to transfer of the filtered hepatocytes to 50 mL centrifuge tubes, the beaker is 725

gently swirled to distribute the hepatocytes evenly. The crude hepatocyte suspension is 726

centrifuged for 3 min at 50 × g, 4°C. 727

42. The gonads (ovaries or testes) are removed in their entirety and weigh to the nearest 728

0.01 g. The gonadosomatic index (GSI) of the donor animal is determined by calculating the 729

gonad weight divided by the whole animal weight (GSI = (100 x the gonad mass) /whole 730

animal mass). Both the gonad weight and GSI are recorded (see Reporting Template). The 731

gonads (testes or ovaries) appear as two strands of tissue that run along the length of the 732

peritoneal cavity on the ventral side of the kidney. Sexual maturity in trout may be 733

determined by the measured GSI. Generally, males with a GSI < 0.05 and females with a GSI 734

< 0.5 may be considered sexually immature. Alternatively, sexual maturity may be 735

determined using histology (9). Detailed descriptions of gonadal development in trout may be 736

found in (10-12). 737

738

43. The supernatant is aspirated to the point where the centrifuge tube begins to taper (~4 739

mL mark) without disturbing the hepatocyte pellet. The supernatant may be aspirated either 740

manually by using a serological pipette, or by using a vacuum pump, but should not be 741

poured. 742

44. Next, 5 mL of perfusion buffer III are added and the hepatocytes are suspended by 743

holding the centrifuge tube at approximately a 60º angle, and gently tapping the bottom of the 744

centrifuge tube on the back of the opposite hand. After visually inspecting the tube for 745

complete hepatocyte suspension (no visible clumps), the final volume is brought up to 32 mL 746

with perfusion buffer III. 747

45. From each 50 mL centrifuge tube, 16 mL of hepatocyte suspension is transferred to a 748

new 50 mL centrifuge tube (so that all tubes contain 16 mL of hepatocyte suspension). To 749

each tube, 14 mL of 90% Percoll solution (4°C or ice) are added and mixed well by gentle 750

inversion. 751

46. The mixture is centrifuged for 10 min at 96 × g, 4°C. The supernatant is immediately 752

removed by aspirating to just above the pellet, and the hepatocytes are suspended in 753

approximately 20 mL of L-15 medium (pH 7.8 at 12°C). Two tubes of hepatocytes are 754

combined into 1. 755

47. After centrifugation of the suspension for 3 min at 50 × g, 4°C to sediment 756

hepatocytes, and re-suspension of the hepatocyte pellet in 20 mL of L-15, two tubes may be 757

combined together into one. Depending upon the number of tubes required for the amount of 758

hepatocytes isolated. This step may be repeated. 759

48. Then the hepatocyte pellet is suspended in 20 – 40 mL of L-15 depending on the 760

expected hepatocyte concentration. All suspensions should be combined into one tube at this 761

point. 762

49. The hepatocyte yield and viability is determined by using a hemocytometer with 763

0.04% trypan blue solution. Hepatocyte counts and viability should be recorded. (see 764

template). 765

50. The total yield from the isolation procedure is calculated as viable hepatocyte 766

concentration (hepatocytes/mL) × suspension volume (prior to cell counting; mL) = total yield 767

21

of hepatocytes. The total number of hepatocytes available for cryopreservation is similarly 768

calculated as viable hepatocyte concentration (hepatocytes/mL) × suspension volume (post-769

cell counting; mL). 770

RT-HEP cryopreservation 771

51. During the entire procedure, the primary hepatocytes and cryopreservation media 772

should be kept on ice unless specifically stated otherwise. 773

52. The pH of all cryopreservation media should be adjusted at the fish maintenance 774

temperature (e.g., 12°C) within 2 h prior to use and maintain on ice or in a 4°C refrigerator. 775

53. The procedure described in the following is designed for 50 cryogenic vials containing 776

1.5 mL of 10 × 106 hepatocytes/mL each (15 × 10

6 hepatocytes per cryogenic vial). Fifty 777

vials will require 750 × 106 hepatocytes, but the number of vials may be scaled up or down 778

depending upon the number of hepatocytes available for cryopreservation. 779

54. Determine the suspension hepatocyte concentration and calculate the volume required 780

for 375 × 106 hepatocytes. Hepatocytes should be concentrated such that the required volume 781

is less than 50 mL (minimum hepatocyte concentration of 7.5 × 106 hepatocytes/mL). 782

375 × 106 hepatocytes / suspension concentration (hepatocytes/mL) = volume required (mL) 783

55. Transfer 375 × 106

hepatocytes into 2 clean 50 mL centrifuge tubes, and bring each 784

tube to a final volume of 50 mL with cryopreservation buffer (see Table 2). 785

56. Centrifuge hepatocytes for 5 min at 50 × g, 4°C to sediment hepatocytes. For each 786

tube, aspirate the supernatant to just above pellet, and then add cryopreservation medium up 787

to 12.5 mL. Assuming no loss of hepatocytes in this step, the concentration will be 30 × 106 788

hepatocytes/mL. 789

57. Suspend hepatocytes by gentle inversion and tapping as described above. Slowly add 790

6.25 mL of cryopreservation medium containing 12% DMSO while gently swirling 791

hepatocytes. 792

58. Maintain the hepatocytes on ice for 5 min, then slowly add 18.75 mL of 793

cryopreservation buffer containing 16% DMSO while gently swirling hepatocytes. The final 794

volume is 37.5 mL at 10 × 106 hepatocytes/mL. 795

59. Maintain the hepatocytes on ice for 5 min, then suspend by gentle inversion. Transfer 796

1.5 mL aliquots of the bulk hepatocyte suspension to the 1.8 mL cryogenic vials. To ensure 797

proper hepatocyte concentration, gently mix or swirl the hepatocytes in the bulk suspension 798

between each transfer. 799

60. Cryopreserve hepatocytes by placing the vials into the vapor phase of liquid nitrogen. 800

61. Store vials in the vapor phase of liquid nitrogen. 801

802

22

REPORTING TEMPLATE 803

804

23

FIGURES 805

806

Figure 1. Set-up for perfusion of a fish liver to obtain primary hepatocytes. Perfusate is 807

pumped first through a water-jacketed coil condenser followed by a water-jacketed bubble 808

trap before perfusing the liver. Water is cooled by a chiller so that the perfusate exiting the 809

bubble trap is maintained at the temperature in which the fish was acclimated. The perfusion 810

line exiting the bubble trap may be split to perfuse two fish, simultaneously (Figure used with 811

permission from Fay et al., 2015 [8]). 812

813

Figure 2. Photograph showing a midline incision from the vent to the isthmus, and a lateral 814

incision extending dorsally from the vent. (Figure used with permission from Fay et al., 2015 815

[8]). 816

817

24

818

Figure 3. Photograph showing the fish’s liver, exposed by cutting away the body wall. The 819

arrow indicates a ventral branch of the hepatic portal vein. (Figure used with permission from 820

Fay et al., 2015 [8]). 821

822

Figure 4. Photograph showing a cannulated liver. The liver will blanch immediately upon 823

insertion of the catheter and perfusion with buffer. Sever the vessels to the heart or the 824

chambers of the heart to allow for perfusate efflux. (Figure used with permission from Fay et 825

al., 2015 [8]). 826

827

25

828

Figure 5. Photograph showing removal of the liver. (Figure used with permission from Fay 829

et al., 2015 [8]). 830

831

Figure 6. Photograph showing the mechanical collection of hepatocytes. (Figure used with 832

permission from Fay et al., 2015 [8]). 833

834

26

835

Figure 7. Photograph showing the crude hepatocyte suspension after filtration through a 836

100μM nylon mesh. (Figure used with permission from Fay et al., 2015 [8]). 837

838

REFERENCES 839

1. Johanning, K., Gauvin, R., Palmer, M., Lehnert, K., Shaw, M., Dungan, L., Whisnart,R., 840

Lohnes, K., and Hill, J. 2010. In vitro rainbow trout (Oncorhynchus mykiss) liver S9 841

metabolism assay: optimizing the assay conditions to determine chemical bioaccumulation 842

potential. SETAC Europe 20th Annual Meeting, Seville, Spain. 843

2. Johanning, K., Hancock, G., Escher, B., Adekola, A., Bernhard, M.J., Cowan-Ellsberry, 844

C., Domoradzki, J., Dyer, S., Eickhoff, C., Embry, M., Erhardt, S., Fitzsimmons, P., 845

Halder, M., Hill, J., Holden, D., Johnson, R., Rutishauser, S., Segner, H., Schultz, I., and 846

Nichols, J. 2012. Assessment of metabolic stability using the rainbow trout 847

(Oncorhynchus mykiss) liver S9 fraction. Current Protocols in Toxicology 53:14.10.1-28. 848



44:345-351. 851

4. Mommsen, T., Moon, T.M., and Walsh, P. 1994. Hepatocytes: isolation, maintenance and 852

utilization. In: Biochemistry and molecular biology of fishes, vol. 3 (P. Hochachka and T. 853

Mommsen, eds.) pp. 355-372. Elsevier, Amsterdam. 854

5. Mudra, D. R. and Parkinson, A. 2001. Preparation of hepatocytes. Current Protocols in 855

Toxicology 8:14.2.1-13. 856

6. Seglen, P. 1976. Preparation of isolated rat liver cells. Methods in Cell Biology 13:29-83. 857

27

7. Segner, H. 1998. Isolation and primary culture of teleost hepatocytes. Comparative 858

Biochemistry and Physiology Part A: 120:71-81. 859




65:4.42.1-29. 863

9. Blazer, V. 2002. Histopathological assessment of gonadal tissue in wild fishes. Fish 864

Physiology and Biochemistry 26:85-1201. 865

10. Billard, R. and Escaffre, A. 1975. Identification of spermatogenesis stages in the rainbow 866

trout based on gonad morphology and spermiation (in French). Bulletin français de la 867

pêche et de la pisciculture 256:111-118. 868

11. Gomez, J.M., Mourot,B., Fostier, A., and Le Gac, F. 1999. Growth hormone receptors in 869

ovary and liver during gametogenesis in female rainbow trout (Oncorhynchus mykiss). 870

Journal of Reproduction and Fertility 115:275-285. 871

12. Le Gac, F., Thomas, J.L., Mourot, B., and Loir, M. 2001. In vivo and in vitro effects of 872

prochloraz and nonylphenol ethoxylates on trout spermatogenesis. Aquatic Toxicology 873

53:187-200. 874

875

28

ANNEX 3 876

CHARACTERIZATION OF RT-HEP 877

1. Freshly isolated RT-HEP should be characterized for % viability, as determined by 878

trypan blue exclusion (see ANNEX 2). RT-HEP thawed from cryopreservation should be 879

characterized for both % viability and % recovery (% viable hepatocytes obtained post-thaw 880

compared to the number cryopreserved, initially) (see ANNEX 5). 881

2. Each hepatocyte lot should be evaluated for the ability to catalyze Phase I and II 882

biotransformation reactions. Assuming the RT-HEP retain their activity upon 883

cryopreservation, ideally these characterization assays should be performed on both freshly 884

isolated and thawed RT-HEP. 885

3. Suggested standardized assays for measuring Phase I and Phase II activity are listed in 886

Table 1 and are briefly described in (1). Table 1 provides an overview of the methods most 887

commonly used, the substrates, and key references. Results from these assays should be 888

included in the report template. 889

4. Activity of biological material may also be evaluated using well-characterized, known 890

test chemicals. 891

5. When applied to RT-HEP, these assays are generally performed on the lysate obtained 892

from a sonicated suspension of hepatocytes. Generally, RT-HEP are diluted to a 893

concentration of 2x106 hepatocytes/mL and briefly sonicated using a hand-held ultrasonic cell 894

disruptor (2), although necessary hepatocyte concentration may change depending on the 895

assay specifications. The results are then normalized to protein content, determined using a 896

commercially available protein assay kit following the manufacturer’s instructions. 897

6. If the likely pathway for biotransformation of a particular test chemical is known, it 898

may be advisable to evaluate this pathway in advance, assuming that a standardized assay for 899

measuring this activity is available. Assays that evaluate endpoints (e.g., enzyme activity at 900

30 min) or activity overtime can be used. If comparing results with other laboratories, it is 901

best to consider the exact conditions (e.g., substrate concentration, protein concentration, 902

incubation time(s), endpoint or overtime incubations) utilized. 903

904

29

Table 1: List of commonly-used enzyme activity assays, substrates, and references that can 905

be used to characterize RT-HEP activity, usually performed with cell lysates. 906

Assay / Activity Enzyme Reaction type Substrate Reference(s)

Phase I

Ethoxycoumarin O-

deethylation (ECOD)

CYP1A O-deethylation 7-Ethoxycoumarin 3,4,5

7-ethoxyresorufin O-

dealkylation (EROD)

CYP1A O-dealkylation 7-Ethoxyresorufin 6

7-methoxyresorufin

O-dealkylation

(MROD)

CYP1A O-dealkylation 7-

Methoxyresorufin

6

7-pentoxyresorufinO-

dealkylation (PROD)

CYP2B O-dealkylation 7-Pentoxyresorufin 6

Testosterone 6β-

hydroxylation

CYP3A Aromatic

ring hydroxylation

Testosterone 7

Chlorzoxazone 6-

hydroxylation

CYP2E1 Aromatic

ring hydroxylation

Chlorzoxazone 8

Lauric acid 11-

hydroxylation

CYP2K1 Long-chain aliphatic

hydroxylation

Lauric acid 6

p-nitrophenyl acetate

hydrolysis

Carboxylesterase Hydrolysis p-nitrophenyl

acetate

9

Phase II

CDNB-glutathione

conjugation

GST Glutathione

conjugation

1-chloro-2,4-

dinitrobenzene

10

p-Nitrophenol

glucuronidation

UGT Glucuronidation p-Nitrophenol 11, 12

907

REFERENCES 908 909










3. Edwards, A.M., Glistak, M.L., Lucus, C.M., and Wilson, P.A. 1984. 7-ethoxycoumarine 919

deethylase activity as a convenient measure of liver drug metabolizing enzymes: 920

Regulation in cultured hepatocytes. Biochemical Pharmacology 33:1537–1546. 921

4. Cravedi, J.P., Perdu-Durand, E., and Paris, A. 1998. Cytochrome P450-dependent 922

metabolic pathways and glucuronidation in trout liver slices. Comparative Biochemistry 923

and Physiology Part C 121:267–275. 924

5. Leguen, I., Carlsson, C., Perdu-Durand, E., Prunet, P., Part, P., and Cravedi, J.P. 2000. 925

Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in 926

culture. Aquatic Toxicology 48:165–176. 927

6. Nabb, D.L., Mingoia, R.T., Yang, C.H., and Han, X. 2006. Comparison of basal level 928

metabolic enzyme activities of freshly isolated hepatocytes from rainbow trout 929

(Oncorhynchus mykiss) and rat. Aquatic Toxicology 80:52–59. 930

30

7. Oesch, F., Wagner, H., Platt, K.L., and Arand, M. 1992. Improved sample preparation for 931

the testosterone hydroxylation assay using disposable extraction columns. Journal of 932

Chromatography 582:232–235. 933

8. Peter, R., Bocker, R., Beaune, P.H., Iwasaki, M., Guengerich, F.P., and Yang, C.S. 1990. 934

Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P-935

450IIE1. Chemical Research in Toxicology 3:566–573. 936

9. Wheelock, C. E., Eder, K. J., Werner, I., Huang, H., Jones, P. D., Brammell, B. F., Elskus, 937

A. A., and Hammock, B. D. 2005. Individual variability in esterase activity and CYP1A 938

levels in Chinook salmon (Oncorhynchus tshawytscha) exposed to esfenvalerate and 939

chlorpyrifos. Aquatic Toxicology 74: 172-192. 940

10. Habig, W.H., Pabst, M.J., and Jakoby, W.B. 1974. Glutathione S-transferases. The first 941

enzymatic step in mercapturic acid formation. Journal of Biological Chemistry 249:7130–942

7139. 943

11. Castren, M., and Oikari A. 1983. Optimal assay conditions for liver UDP-944

glucuronosyltransferase from the rainbow trout, Salmo gairdneri. Comparative 945

Biochemistry and Physiology Part C 76:365–369. 946

12. Ladd, M.A., Fitzsimmons, P.N., and Nichols, J.W., 2016. Optimization of a UDP-947

glucuronosyltransferase assay for trout liver S9 fractions: activity enhancement by 948

alamethicin, a pore-forming peptide, Xenobiotica, DOI:10.3109/00498254.2016.1149634. 949

950

951

31

ANNEX 4 952

HEAT-INACTIVATION OF RT-HEP 953

1. It is suggested that laboratories consider preparing a large volume of enzymatically 954

inactive RT-HEP in advance, and freeze them as aliquots (e.g., 5 mL). 955

2. Active RT-HEP unused at the end of a test may become material for subsequent heat-956

inactivation. 957

3. Equipment: 958

− Hot plate 959

− Beaker with water (water bath) 960

− Vessel for boiling the cells within the water bath 961

− Graduated cylinder 962

963

4. The RT-HEP suspension should be diluted in L-15 medium to the desired 964

concentration for test use (e.g., 2 ×106 hepatocytes/mL). When using cryopreserved RT-HEP, 965

an abbreviated thawing protocol may be sufficient. 966

5. The volume of the suspension should be recorded and the suspension transferred to a 967

heat-safe vessel (preferably glass). 968

6. A beaker of water is heated on a hotplate and the water brought to boiling. The vessel 969

with the RT-HEP suspension is placed into the boiling water bath, and the suspension brought 970

to a slow boil for 15 min. 971

7. After the suspension has cooled, the suspension is transferred to a graduated cylinder 972

and the volume adjusted by adding L-15 medium or water to maintain the desired hepatocyte 973

concentration. 974

8. The enzymatically inactive RT-HEP suspension is stored at -20 or -80°C. 975

976

32

ANNEX 5 977

THAWING OF CRYOPRESERVED RT-HEP 978

1. Cryopreserved RT-HEP should be thawed following the instructions of the supplier. 979

2. If the RT-HEP were isolated and cryopreserved according to the protocol given in 980

ANNEX 2, the procedure described here should be followed to thaw the RT-HEP. Thawing 981

of the cryopreserved RT-HEP can be expected to result in a 25-45% yield (compared to the 982

number of hepatocytes frozen) with high viability (>85%) (1, 2, 3, 4). 983

Apparatus and Material 984

3. The following apparatus and material is needed: 985

Refrigerated centrifuge (e.g., for 50 mL tubes) 986

Conical centrifuge tubes, e.g., 50 mL 987

Water bath (room temperature) 988

1000 µL pipette with tips 989

Serological pipets (2.5 - 25 mL) and pipet-aid 990

Cryogenic vials containing 1.5 mL of cryopreserved RT-HEP at 10 ×106 cells/mL 991

1.5 mL micro-centrifuge tubes and rack 992

Sterile vacuum filters (0.2 µm polyethersulfone (PES) membrane) 993

Recovery medium (see Table 1) 994

Dulbecco’s Modified Eagle Medium (DMEM), low glucose with phenol red 995

Fetal Bovine Serum (FBS), non heat-inactivated 996

Bovine Serum Albumin (BSA) 997

Leibovitz L-15 medium (L-15) with glutamine, without phenol red 998

Trypan blue, 0.4% 999

1 N NaOH, and HCL (for pH adjustment) 1000

Preparation of media 1001

Determination of the total number of RT-HEP and volume of media needed 1002

4. Assuming a 25-45% yield (§2), 2 vials containing 1.5 mL of 10 ×106 cells/mL 1003

suspension each can be expected to provide ~ 7.5 – 13.5 × 106 viable RT-HEP. 1004

5. The total number of cryogenic vials needed should be determined to provide enough 1005

RT-HEP taking into account that the substrate depletion method described in this Test 1006

Guideline is typically conducted at 1-2 ×106 cells/mL. 1007

6. It is recommended that the user thaw 2-3 vials together in one 50 mL tube with 1008

recovery medium for superior yield. Thawing 1 vial alone may result in <25% recovery from 1009

cryopreservation. 1010

7. The volume of recovery medium should be determined assuming that 44-45 mL of 1011

recovery medium are needed for one tube (see below). The total volume of L15 medium can 1012

be determined calculating a volume of ~100 mL per tube. 1013

1014

33

Recovery medium preparation (prior the test day) 1015

8. The recovery medium should be prepared prior to the test day using the ingredients 1016

provided in Table 1. The medium is sterile filtered and may need to be pH adjusted. Allow 1017

the solution to sit overnight at 1-10°C to reduce foam. 1018

Table 1: Recovery medium 1019

Reagent Per 100 mL

preparation Final concentration

DMEM 90 mL

FBS 10 mL 10% (v/v)

BSA 0.25 g 0.25% (w/v)

1020

Media preparation (test day) 1021

9. On the day of the test, the pH of the recovery medium should be re-adjusted to 7.8 1022

±0.1 at 10-15°C. 1023

10. 42 mL of recovery medium should be added into each 50 mL tube. An additional tube 1024

with recovery medium for cryovial washes (see below) should be prepared. The recovery 1025

medium is kept at room temperature. The pH of ca. 100-150 mL L-15 medium is adjusted to 1026

pH 7.8 at 10-15°C using 1 N HCl or NaOH. L-15 is kept on ice. 1027

Thawing procedure 1028

11. The cryogenic vials are removed from the liquid nitrogen vapour and immediately 1029

placed in a room temperature water bath, holding them by their caps above the water level so 1030

that the frozen suspensions are below the water line. The vials are gently moved side to side 1031

until contents freely move and a small ice crystal remains. Typically, the thawing process 1032

takes about 2 min 15 sec. If the location of the cryopreserved RT-HEP and the laboratory 1033

used for thawing are different, it is recommended that the user transport the vials on dry ice. 1034

12. The content of 2-3 vials is poured into one 50 mL centrifuge tube containing 42 mL of 1035

recovery medium (room temperature). 1036

13. Emptied cryovials are rinsed with recovery medium to suspend any remaining RT-1037

HEP: 1 mL of recovery medium from the extra tube is added to the empty cryovials, the 1038

cryovials are then capped and inverted once to mix. The contents from the rinse are added to 1039

the 50 mL tube. 1040

14. The centrifuge tubes are capped and gently inverted, followed by centrifugation for 5 1041

min at 50 × g, 4°C. 1042

15. The supernatant should be aspirated to the point where the centrifuge tube begins to 1043

taper (around 4 mL), being careful not to disturb the cell pellet. Aspirating close to the pellet 1044

may decrease yield. To obtain consistent results, the supernatant should be aspirated to the 1045

conical portion of the tube for all wash steps. The supernatant can be aspirated manually by 1046

using a pipette, or by using a vacuum pump. It should not be discarded by pouring. 1047

34

16. 5 mL of L-15 medium (pH 7.8 ±0.1, 4°C or ice cold) is added to the centrifuge tube 1048

and the cell pellet suspended. This should be done gently by tapping the side of the centrifuge 1049

tube against the back of the opposite hand. Suspensions from 2 tubes are combined into 1, if 1050

applicable, and all tubes are brought to a final volume of 45 mL with the L-15. The tubes are 1051

capped and inverted once and centrifuged at 50 × g for 5 min at 4°C. 1052

17. Steps described in 12 & 13 are repeated if more vials are thawed and used. Again, 1053

RT-HEP are combined into 1 centrifuge tube, if applicable, and add L-15 to a final volume of 1054

45 mL. 1055

18. The tube is capped and inverted once and centrifuged at 50 × g for 3 min at 4°C. 1056

19. The supernatant is aspirated to just below (~2 mm) the conical portion of the 1057

centrifuge tube, and approximately 0.75 mL of L-15 is added per cryogenic vial thawed. The 1058

RT-HEP pellet is suspended. 1059

20. The volume of suspension is measured by using a serological pipet of appropriate size. 1060

21. The viable and dead RT-HEP are counted using a hemocytometer and 0.04% trypan 1061

blue and dilute to the desired cell concentration) as described in (5). Alternative methods to 1062

count cells may be used (e.g., cell counters). 1063

22. The % of RT-HEP recovery from cryopreservation is calculated based on the volume 1064

of the cell suspension (see §19 above) and cell count results. 1065

Number of

recovered

(viable) RT-HEP

= Average viable RT-HEP

concentration x

Suspension volume prior to RT-HEP

counting

1066

% RT-HEP

recovery

= 100 x (Number of recovered RT-HEP /

Number of viable RT-HEP initially

cryopreserved)

1067

23. The RT-HEP suspension may be diluted to the desired incubation concentration by 1068

adding an appropriate volume of L-15 medium. 1069

24. The RT-HEP suspension should be kept on ice until use. 1070

1071

REFERENCES 1072



44:345-351. 1075





35

1080

3. Markell, L.K., Mingoia, R.T., Peterson, H.M., Yao, J., Waters, S.M., Finn, J.P., Nabb, 1081

D.L., and Han, X. 2014. Endocrine disruption screening by protein and gene expression of 1082

vitellogenin in freshly isolated and cryopreserved rainbow trout hepatocytes. Chemical 1083

Research in Toxicology 27:1450-1457. 1084

4. Mingoia, R.T., Glover, K.P., Nabb, D.L., Yang, C.-H., Snajdr, S.I., and Han, X. 2010. 1085

cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss): A validation study 1086

to support their application in bioaccumulation assessment. Environmental Science & 1087

Technology 44:3052-3058. 1088




65:4.42.1-29. 1092

1093

36

ANNEX 6 1094

PRELIMINARY EXPERIMENTS TO ESTABLISH REACTION CONDITIONS 1095

1. The primary goal of conducting preliminary experiments is to determine reaction 1096

conditions that result in first-order depletion kinetics. These experiments are used to establish 1097

a sampling schedule that captures the depletion of the test chemical (as significantly different 1098

from the negative controls) while preserving the ability to quantify the test chemical 1099

concentration in the system at final points. Preliminary experiments are performed with 1100

biological material (i.e. cryopreserved RT-HEP) that has been characterized for Phase I and II 1101

metabolic enzyme activity (refer to ANNEX 3). 1102

2. To obtain a chemical depletion rate for use in the in vitro to in vivo extrapolation 1103

model, it is generally desirable to achieve 20% to 90% depletion of the test chemical over the 1104

course of a test. Variables that can be tested to achieve this goal include: the concentration of 1105

RT-HEP, starting test chemical concentration, and total incubation time (1). 1106

3. In addition to these preliminary experiments, other considerations for achieving an 1107

accurate substrate depletion measurement include: sensitivity of the analytical method and the 1108

need for an internal standard, solvent selections to dissolve the test chemical, introduction into 1109

the system and reaction termination, and the use of positive (reference chemical) and negative 1110

controls (see ANNEX 4 and [2]). 1111

4. It is generally recommended that substrate depletion experiments, including 1112

preliminary experiments should be conducted at concentrations of 1-2.0 x 106 1113

hepatocytes/mL. 1114

5. The test chemical concentration should not demonstrate cytotoxicity. Therefore, 1115

viability of RT-HEP exposed to the test concentration should be evaluated, e.g., by using 1116

trypan blue exclusion. RT-HEP viability should be ≥ 80%. 1117

6. The starting test chemical concentration is determined by the need to achieve first-1118

order kinetics as well as the sensitivity of the analytical method, keeping in mind the possible 1119

need to measure concentrations substantially lower than starting values (i.e., at later time 1120

points). The sensitivity of the analytical method should be able to accurately measure all time 1121

points or 10% of the initial test chemical concentration. Theory dictates that the likelihood of 1122

first-order kinetics increases as the starting concentration is decreased below the Michaelis-1123

Menten constant, KM. The KM is the substrate concentration at which the reaction rate is ½ 1124

Vmax (maximum rate achieved by the system at substrate maximum saturation concentration). 1125

Realistically, it is not always possible to achieve these concentrations due to detection 1126

limitations of the analytical method for the test chemical. Practical experience suggests that a 1127

starting concentration in the very low micromolar/high nanomolar range (e.g., ≤ 1.0 μM) 1128

often yields satisfactory results, although users should try to perform depletion experiments at 1129

the lowest reasonable test chemical concentration. Further discussion of chemical 1130

concentrations is included in the Guidance Document. 1131

7. In order to choose appropriate starting test chemical concentration, three 1132

concentrations may need to be evaluated: a) 1.0 μM or other concentration based on previous 1133

information, if available; b) lowest concentration-quantifiable assuming 50% depletion, and c) 1134

a concentration in between a) and b). 1135

37

8. A decision tree may be employed to make the final decisions regarding the starting 1136

incubation concentration for each test chemical. 1137

1138

1139

1140 1141

1142

9. Preliminary experiments are generally conducted with a limited number of time points 1143

(e.g., 0.1, 1 and 2 h) and replication (e.g., one duplicate per time point). The initial test 1144

chemical concentration resulting in the most rapid depletion rate is usually preferred for the 1145

definitive study. If several test chemical concentrations generate similar depletion rates, the 1146

higher concentration is preferred for the definitive study as it minimizes detection limit 1147

challenges. 1148

10. Depending on the need, the sampling scheme may span <10 min up to 4 h or more in 1149

few instances incorporating the recommended six or more individual sampling time points. 1150

11. The net result is that first-order depletion rate constants derived from these tests can 1151

not be expected to vary in direct proportion to the cell density or test chemical concentration. 1152

Very low rates of biotransformation can be addressed by increasing the incubation time up to 1153

4 h. Increasing the RT-HEP number or density above 2 x 106

cells/ml to promote increased 1154

levels of activity is not recommended to avoid saturation of enzymes. 1155

12. A departure from first-order kinetics can be expected if the starting chemical 1156

concentration saturates the activity of enzymes responsible for chemical clearance. For 1157

reaction pathways that exhibit classical Michaelis-Menten kinetics, this saturation will result 1158

in zero-order elimination. The appearance of zero-order kinetics suggests that the starting 1159

chemical concentration should be reduced (3). 1160

13. Alternatively, log-transformation of the data may yield a pattern suggesting bi-1161

exponential kinetics with an initial “fast” depletion phase followed by a “slow” terminal 1162

Do the rates vary with concentration?

NO – choose the historical value.

YES – Do the rates of the lowest 2

concentrations vary?

YES – Choose the lowest concentration

which can be comfortably analyzed.

NO – Choose the higher of the two concentrations.

38

depletion phase. This pattern can be caused by product inhibition, wherein the accumulation 1163

of metabolic products inhibits enzymatic activity at later time points, cofactor limitation or 1164

enzyme saturation Reduction of both the starting chemical concentration and cell 1165

concentration may be attempted in an effort to eliminate this problem (1). 1166

1167

REFERENCES 1168




65:4.42.1-29. 1172

2. OECD xxxx. Guidance document No xx Determination of in vitro intrinsic clearance 1173

using cryopreserved hepatocytes (RT-HEP) or liver S9 sub-cellular fractions (RTL-S9) 1174

from rainbow trout and extrapolation to in vivo intrinsic clearance. 1175

1176






1182

39

ANNEX 7 1183

1184

TEST SET-UPs 1185 1186

Test Set-up 1: Single vial approach (Figure 1) 1187

1. The single vial approach is recommended to test chemicals that are not difficult-to-test 1188

(e.g., non-volatile, does not bind to vessel walls, and distributes rapidly through the reaction 1189

system). It generally produces the least variable results and is simplest to perform. 1190

2. As described in §29ff of the main text, incubations are carried out in a single vial 1191

containing e.g., 1 mL of RT-HEP suspension. Samples (100 µL) are taken at the defined time 1192

points from this vial and are transferred into a micro-centrifuge tube containing stopping 1193

solution. 1194

3. A minimum number of 6 time points is required to determine the CL, IN VITRO, INT rate; 1195

therefore, the test set-up should include ≥6 time points. 1196

1197

Figure 1. Test Set-up 1: Independent runs using the single vial approach 1198

1199

40

Test Set-up 2: Multiple vial approach (Figure 2) 1200

4. This set-up is recommended for volatile test chemicals. 1201

5. Incubations with volatile test chemicals can be performed by sealing the vials 1202

containing e.g., 200 μL of RT-HEP suspension with a septum-lined cap after the pre-1203

incubation period. A syringe is then used to introduce both the test chemical and stopping 1204

solution. As for the single-vial approach, east test consists of at least two independent runs to 1205

determine the CL, IN VITRO, INT rate. Each independent run is performed on a different day or 1206

on the same day, provided that for each run: a) independent fresh stock solutions and 1207

working solutions of the test chemical area prepared and; b) independently prepared RT-HEP 1208

suspensions are used. For each run, the pre-determined number of vials is prepared for active 1209

RT-HEP (e.g., total of 14, 7 for test chemical and 7 for the reference chemical) and for 1210

enzymatically inactive RT-HEP (e.g., 7). The vials are spiked with the test chemical and the 1211

reference chemical as shown in Figure 2. Stopping solution is added directly to each sample 1212

vial at the various time points (e.g., 5, 10, 15, 20, 40, 60, 90 min). Additional details are 1213

provided in (1). 1214

1215

Figure 2. Test Set-up 2: Independent runs using the multiple vial approach 1216

1217

REFERENCES 1218




65:4.42.1-29. 1222

draft oecd test guideline determination of in vitro

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